| Craniopharyngioma(CP) accounts for approximately1.2%~4.6%of all intracranialtumors. Their close proximity to vital structures such as the hypothalamic-pituitary axisand optic apparatus makes them one of the most challenging and controversialmanagement dilemmas in neurosurgery. Recurrence following initial transcranial resectionis reported as9%~51%at a median time of26months to96months[1-3]. Treatment optionsfor recurrent CP include repeat surgery, radiotherapy, radiosurgery and intracystictherapies[4-7]. Application of32P colloid for radiotherapy on the treatment of cystic CP bystereotactic surgery is one of characteristic clinical project in neurosurgery department ofnavy general hospital[8-12]. At present there are tens of thousands of patients besuccessfully treated by the medical project at home and abroad[13-16], but there were stillindividual patients died, postoperative visual acuity and visual field damaged, pituitaryfunction changed, etc[17-19].32P colloid is radioactive and toxicity, many clinical problemsto be solved: whether cerebrospinal fluid infiltrated during intraoperative, the range of drugdistribution, the drug drainage pathway and residue pollution postoperative, the cysticcavity volume shrinking, the changes of blood tests, the specific mechanism of32Ptreatment to CP cells, the relationships of dose effect and time effect and so on. All need tostrengthen clinical assessment and basic research, so as to provide experimental basis forthe choice of operation, individualized treatment plan and management of postoperativepatients. Therefore, the following three parts were studied respectively.Part I Drug distribution and clinical safety studies on the radiotherapy of32Pcolloid to cystic craniopharyngioma by stereotactic surgeryObjective: Research on how different doses and cystic cavity volume of32P colloid toinfluence the leakage and drug distribution inside the capsule and the clinical safety ofpatients. To detect changes of postoperative blood and urine Phosphorus-32pollution,postoperative pituitary hormone, routine blood, clotting, hepatic and renal function indexes,so as to provide clinical guidance of surgical puncture path selection and operation skillscontrol, dose control, postoperative management for patients.Patients and Methods: By using prospective study method, from March1,2012toDecember31,2013, the first onset or recurrence40patients with cystic CP in neurosurgerydepartment of navy general hospital were divided into4groups according to thetherapeutic dose(milli Curie, mCi):0.5mCi group(10cases),1mCi group(10cases), 1.5mCi group(10cases) and2mCi group(10cases). After32P colloid with Iopamidol300injection diluted by1:1proportion, the mixture was injected into capsule for surgicaltreatment,2h after injection for head CT scan, to observe the postoperative drugdistribution and leakage, to compare difference between groups. Furthemore,24patientsform above were divided into three groups according to the therapeutic dose:0.5mCigroup(8cases),1mCi group(8cases),2mCi group(8cases). To compare respectivelypatient’s preoperative and postoperative visual acuity, visual field and fundus, the results ofhead enhanced MRI or CT, the indexes of routine blood, clotting, electrolyte, hepatic andrenal function, the levels of pituitary hormone. To measure patient’s32P colloid radioactivecount per minute(CPM) of blood and urine by CAPRAC well type NaI gamma counter in1,3and7days after surgery to judge whether there is residual pollution.Results: There were6cases(60.00%) in2mCi group,2cases(20.00%) in1.5mCigroup,1case(10.00%) in1mCi group occurred uneven distribution of drug andcerebrospinal fluid leakage inside capsule for puncture pathway in the brain can’t avoid theventricle, the rest of the patients were well-distributed and no leakage. The comparisonamong groups of numerical difference between preoperative with7days postoperative of24patients’ routine blood indexes, blood biochemical indexes, pituitary hormone levelshad no statistical significance(P>0.05). But there were1case occurred postoperativereduced visual acuity and2cases of postoperative transient diabetes insipidus in2mCigroup. There was1case occurred postoperative transient diabetes insipidus in1mCi group.Patients recovered well in0.5mCi group. There were2cases occurred postoperativeabnormal of hepatic function,1case of coagulation function abnormality,3cases ofelectrolyte disorder and3cases of pituitary hormone levels abnormalities. Thepostoperative1d blood CPM value of2mCi group and1mCi group compared with controlgroup was statistically difference(P<0.05). The postoperative1d,3d,7d urine CPM valueof2mCi group and1mCi group compared with control group was statisticallydifference(P<0.05). Blood pollution returned to normal within3days but urine pollutiondropped slowly close to normal in1week after surgery.Conclusion: The biger of the craniopharyngioma cystic cavity volume, the greaterneed to32P colloid dose, the higher incidence of the puncture pathway through brainventricle and more easily to leakage. So, we should pay attention to prevent intraoperativecerebrospinal fluid leakage of ventricle, monitor blood and urine pollution postoperation,strengthen to monitor abnormal changes of vision and visual field, pituitary hormone levels, blood indexes, and to extend the time of postoperative follow-up.Part II Establish craniopharyngioma limited cell line in vitroObjective: To establish an efficient method for primary culture and subcultureadamantinomatous CP cells and to observe the growth and biological characteristics ofcells in vitro.Materials and Methods: To13cases of adamantinomatous CP fresh tissue samplesconfirmed by pathology, this study pruned organization using microscopic, digested cellsusing pancreatic enzyme by the method of different speed adherence and used the methodof mechanical shave off to establish craniopharyngioma cell line. The cell growth and cellmorphology was observed by inverted microscope and hematoxylin-eosin(HE) staining.The cell ultrastructure was observed by transmission electron microscope(TEM). Theexpression of pan cytokeratin(Pan-CK) was examined by immunohistochemical staining.This study used methyl thiazolyl tetrazolium(MTT) assay to draw the cell growth curveand used flow eytometry to analyze the cell cycle.Results:13cases of CP tissue purified and original generation cultured successfully9cases(69.23%), subcultured successfully7cases(53.85%). The oldest cells survived for7generations and63days. The second generation CP cells grew more stable and survivedlonger time than the sixth generation. The characteristics of second generation CP cellswere typical epithelioid cells and diploid cells, cell cycle mainly in G0-G1and G2-Mperiod.Conclusion: Adamantinomatous CP cells primary purification cultured successfullyand can continuous subcultured limited. Its survival and growth characteristics can meetthe requirements of further processing experiment.Part III Experimental study on32P colloid induce apoptosis ofcraniopharyngioma cells in vitroObjective: Study on whether32P colloid inducing CP cells apoptosis in vitro, therelationships of concentration effect and time effect.Materials and Methods: This study established CP limited cell line by primary cellculture. This study used MTT assay to draw the cell survival rate curve after CP cellstreated by32P colloid with different concentration and different time. This study detectedapoptosis rate by flow cytometry, apoptosis deoxyribonucleic acid(DNA) feature byterminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling(TUNEL)fluorescent staining, apoptosis cell form by Hoechst33342fluorescence staining, apoptosis cell ultrastructure by TEM.Results: Hoechst33342fluorescence staining, TUNEL fluorescence staining andTEM all confirmed that32P colloid did cause the CP cells apoptosis.32P colloid reduced CPcells survival rate and increased apoptosis rate with the increase of concentration(0~14.8MBq/ml) and time(1~14d).Conclusion:32P colloid can effectively inhabit CP cells growth and induce apoptosisin vitro. The higher concentration and longer treatment time induced the more obviouseffect.In conclusion, the research studied drug distribution and clinical safety on theradiotherapy of32P colloid to cystic CP by stereotactic surgery first, and then successfulprimary cultured CP tissue, established and identified the limited adamantinomatous CPcell line in vitro. The research also found that32P colloid can inhibit CP cells growth byway of inducing apoptosis. These results provide guidance for the selection of surgicalpuncture path, operation techniques and dose control, management of patients after sugery.As well as, the research provided foundations for experiment studies on transplanted tumorin nude mice and its angiogenesis effect induced by32P colloid. |
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